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Bacillus licheniformis is recognised as an exceptional expression platform in biomanufacturing due to its ability to produce high-value products. Consequently, metabolic engineering of B. licheniformis is increasingly pursued to enhance its utility as a biomanufacturing vehicle. Effective B. licheniformis cell factories require promoters that enable regulated expression of target genes. This review discusses recent advancements in the characterisation, synthesis, and engineering of B. licheniformis promoters. We highlight the application of constitutive promoters, quorum sensing promoters, and inducible promoters in protein and chemical synthesis. Additionally, we summarise efforts to expand the promoter toolbox through hybrid promoter engineering, transcription factor-based inducible promoter engineering, and ribosome binding site (RBS) engineering. [ABSTRACT FROM AUTHOR]

Catabolite control protein A (CcpA) is a critical regulator in Gram-positive bacteria that orchestrates carbon metabolism by coordinating the utilization of different carbon sources. Although it has been widely proved that CcpA helps prioritize the utilization of glucose over other carbon sources, this global regulator's precise mechanism of action remains unclear. In this study, a mutant Bacillus licheniformis deleted for CcpA was constructed. Cell growth, carbon utilization, metabolites and the transcription of key enzymes of the mutant strain were compared with that of the wild-type one. It was found that CcpA is involved in the regulation of glucose concentration metabolism in Bacillus. At the same time, CcpA regulates glucose metabolism by inhibiting acetic acid synthesis and pentose phosphate pathway key gene zwF. The conversion rate of acetic acid is increased by about 3.5 times after ccpA is deleted. The present study provides a new mechanism of carbon metabolism and acetic acid balance regulated by CcpA. On the one hand, this work deepens the understanding of the regulatory function of CcpA and provides a new view on the regulation of glucose metabolism. On the other hand, it is helpful to the transformation of B. licheniformis chassis microorganisms. [ABSTRACT FROM AUTHOR]

Amino acid efflux and influx transport systems play vital roles in industrial microorganisms' cell growth and metabolism. However, although biochemically characterized, most of them remain unknown at the molecular level in Bacillus licheniformis. In this study, three proteins, namely, YdgF, YvbW, and YveA, were predicted to be involved in the active transport of L-aspartate (L-Asp). This was verified by manipulating their encoding genes. When growing in the minimal medium with L-Asp as the only carbon and nitrogen source, the growth of strains lacking proteins YdgF, YvbW, and YveA was significantly inhibited compared with the wild-type strains, while supplementing the expression of the corresponding proteins in the single-gene knockout strains could alleviate the inhibition. Upon overexpression, the recombinant proteins mediated the accumulation of L-aspartate to varying degrees. Compared with the wild-type strains, the single knockout strains of the three protein genes exhibited reduced absorption of L-aspartate. In addition, this study focused on the effects of these three proteins on the absorption of β-alanine, L-glutamate, D-serine, D-alanine, and glycine. [ABSTRACT FROM AUTHOR]

Bacillus licheniformis is a significant industrial microorganism. Traditional gene editing techniques relying on homologous recombination often exhibit low efficiency due to their reliance on resistance genes. Additionally, the established CRISPR gene editing technology, utilizing Cas9 endonuclease, faces challenges in achieving simultaneous knockout of multiple genes. To address this limitation, the CRISPR-Cpf1 system has been developed, enabling multiplexed gene editing across various microorganisms. Key to the efficient gene editing capability of this system is the rigorous screening of highly effective expression elements to achieve conditional expression of protein Cpf1. In this study, we employed mCherry as a reporter gene and harnessed P mal for regulating the expression of Cpf1 to establish the CRISPR-Cpf1 gene editing system in Bacillus licheniformis . Our system achieved a 100 % knockout efficiency for the single gene vpr and up to 80 % for simultaneous knockout of the double genes epr and mpr . Furthermore, the culture of a series of protease-deficient strains revealed that the protease encoded by aprE contributed significantly to extracellular enzyme activity (approximately 80 %), whereas proteases encoded by vpr , epr , and mpr genes contributed to a smaller proportion of extracellular enzyme activity. These findings provide support for effective molecular modification and metabolic regulation in industrial organisms., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors.)

Background: Robot-assisted technology has been widely used in orthopedic surgery, which can provide surgeons with higher accuracy and reduce radiation exposure. In spinal surgery, robots are often used to assist pedicle screw implantation, while there are relatively few studies on robot-assisted percutaneous kyphoplasty (PKP) under local anesthesia., Methods: A total of 96 patients with single-segment OVCF who met the inclusion criteria were included in this study. Fifty-six patients underwent robot-assisted PKP and forty patients underwent conventional PKP by the same group of surgeons. Collect the relevant parameters., Results: The puncture time and fluoroscopy times during puncture in the robot group were significantly less than those in the manual group ( P  < 0.001). The success rate of first puncture in the robot group was 92.5%., Conclusions: PKP under local anesthesia assisted by the new spinal surgical robot effectively reduces the patient's intraoperative discomfort and has a low learning curve., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Xue, Liu, Li, Xiao, Zhu, Wu and Zhang.)

Malate is an essential intermediate in the tricarboxylic acid (TCA) cycle; it also has valuable uses in medicine and food. The production of malate with a microbial synthesis method is still in its early stages. One of the key problems in metabolic engineering is that the dynamic and subtle changes in malate are difficult to detect. It remains critical to develop techniques with direct and precise detection of malate in microbial metabolism, which facilitates high-throughput screening of the engineered strains. In this study, a genetically encoded biosensor in response to malate was constructed in B. licheniformis . Key regulator MalR and the action site of the biosensor were first identified. Then, the output of the reporter gene expression was amplified by introducing a strong constitutive promoter and iteratively tuning the action sites. The engineered biosensor can respond to malate from 5 to 15 g/L; within this range, it shows a linear correlation between eGFP fluorescence and malate concentration. This biosensor enrich our toolbox of synthetic biology in pathway engineering for malate production in microorganisms.